This continuation application (FIRST Award conversion to RO1) will further explore the neuroanatomical substrates mediating the early stages in the development of opioid dependence. Withdrawal precipitated by an opioid antagonist after a single treatment with morphine reveals the formation of rapid neuroadaptive responses to opioids (acute dependence). Work completed in the prior funding period established quantitative animal models of acute opioid dependence, and revealed that brain systems mediating affective components of opioid withdrawal ("extended amygdala" reward systems) undergo a more rapid and robust adaptive response to acute morphine than do systems mediating somatic components of withdrawal. Additional studies identified an important contribution of associative conditioning mechanisms to the progressive increase in withdrawal severity produced by repeated morphine treatments. Studies proposed herein will further explore the role of individual components of reward circuitry in the direct (unconditioned) components of withdrawal from acute morphine, and in the formation of conditioned associations between environmental stimuli and the acute withdrawal response. Unconditioned withdrawal signs to be measured will include suppression of operant responding for food, brain stimulation reward threshold elevations, anxiogenic-like behavior in the elevated plus-maze, and somatic signs, all precipitated by opioid antagonists following single or repeated intermittent treatment at weekly intervals with a low dose of morphine. Initial studies (Specific Aim 1) will utilize local intracerebral application of the lipophobic antagonist methylnaloxonium (MNX) to identify specific components of the "extended amygdala" wherein opioid receptor blockade results in individual behavioral measures of withdrawal. Specific Aim 2 will employ the same approach to identify the critical neural substrates wherein opioid receptor blockade supports the formation of conditioned associations between precipitated withdrawal and novel environmental stimuli, using the conditioned place aversion paradigm. In Specific Aim 3, functional inactivation of cortical and limbic afferents to the "extended amygdala" through local microinjection of lidocaine will identify circuits critical in the acquisition and expression of conditioned place aversion. Understanding of CNS systems mediating unconditioned and conditioned withdrawal in the early development of opioid dependence should provide information critical for prevention and treatment of opioid addiction, with particular emphasis on the role of acute dependence in the transition from casual drug use to loss of control and compulsive use